Tetanus toxin binds neuronal tissue prior to internalization and trafficking to the central nervous system. Binding of the carboxy‐terminal 50 kDa HC fragment of tetanus toxin to polysialogangliosides is important for this initial cell binding step. Using the three‐dimensional structure of HC, mutants were designed to investigate the role of individual residues in ganglioside binding. Mutant proteins were tested for binding to GT1b gangliosides, to primary motoneurons and for their ability to undergo retrograde transport in mice. Two classes of mutant were obtained: (i) those containing deletions in loop regions within the C‐terminal β‐trefoil domain which showed greatly reduced ganglioside and cell binding and did not undergo retrograde transport and (ii) those that showed reduced ganglioside binding, but retained primary neuronal cell binding and retrograde transport. The second class included point mutants of Histidine‐1293, previously implicated in GT1b binding. Our deletion analysis is entirely consistent with recent structural studies which have identified sugar‐binding sites in the immediate vicinity of the residues identified by mutagenesis. These results demonstrate that ganglioside binding can be severely impaired without abolishing cell binding and intracellular trafficking of tetanus toxin.

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The nontoxic binding domain of tetanus toxin (fragment C or TTC) readily undergoes retrograde axonal transport from an intramuscular injection site. This property has led to investigation of TTC as a possible vector for delivering therapeutic proteins to motor neurons. However, the vast majority of individuals in the developed world have been vaccinated with tetanus toxoid and have circulating antitetanus antibodies that cross-react with TTC and may block the delivery of a TTC-linked therapeutic protein. However, it is uncertain whether the immune response is capable of completely neutralizing an intramuscular depot of protein prior to its internalization by presynaptic nerve terminals, where it is inaccessible to antibody. We have evaluated uptake of rhodamine-labeled TTC following intramuscular injection in normal animals and animals vaccinated with tetanus toxoid prior to injection of fluorescently labeled TTC. All animals demonstrated uptake of TTC, with fluorescence appropriately localized to the hypoglossal nerve and nucleus. The distribution and intensity of fluorescence within neurons and processes were indistinguishable between the two groups and were characteristic of TTC. Vaccinated animals showed levels of uptake of TTC into the brain comparable to those of immunologically naïve animals as measured by quantitative fluorimetry. All vaccinated animals had protective levels of antitetanus antibodies as measured by ELISA. Uptake of TTC by nerve terminals from an intramuscular depot is an avid and rapid process and is not blocked by vaccination associated with protection from tetanus toxin.

One goal of gene therapy is the targeted delivery of therapeutic genes to defined tissues. One attractive target is the central nervous system as there are several neuronal degenerative diseases which may be amenable to gene therapy. At present there is a lack of delivery systems that are able to target genes specifically to neuronal cells. Multi-domain proteins were designed and constructed to facilitate the delivery of exogenous genes to neuronal cells. Neuronal targeting activity of the proteins was achieved by inclusion of the HC fragment of tetanus toxin (TeNT), a protein with well-characterised tropism for the central nervous system. The yeast Gal4 DNA-binding domain enabled specific binding of DNA while the translocation domain from diphtheria toxin (DT) was included to facilitate crossing of the endosomal vesicle. One multi-domain protein, containing all three of these domains, was found to transfect up to 8% of neuroblastoma N18-RE105 cells with marker genes. Monitoring the transfection by confocal microscopy indicated that this protein-DNA transfection complex is to some extent localised at the cell surface, suggesting that further improvements to translocating this membrane barrier may yield higher transfection levels. The demonstration that this multi-domain protein can target genes specifically to neuronal cells is a first step in the development of novel vectors for the delivery of genes with therapeutic potential to diseased neuronal tissues.